It was demonstrated previously that macrophages specifically interact with a distinct B cell subpopulation which is characterized as Lyb5+. Current experiments have demonstrated that Lyb5- B cells can be stimulated by the mitogen LPS. To gain further insights into the activation requirement of B cells which comprise the Lyb5- B cell subpopulation, the ability of lipoprotein free (phenol extracted) and lipoprotein rich (butanol extracted) LPS to stimulate Lyb5- B cells was examined. We used the bromodeoxyuridine (BUdR) + light technique to specifically eliminate B cells which respond to one type of LPS and determine whether the remaining B cells can respond to the other LPS extract. This approach allowed us to identify in normal mice a subset of B cells which respond to butanol-extracted LPS but not to pheno-extracted LPS. A similar subset was found in neonatal (less than 2 weeks old) mice which have not yet developed Lyb5+ B cells. Thus, the pool of Lyb- B cells in normal mice can be divided into two subsets: one which responds both to pheno-extract and to butanol-extract LPS, and a separate cell pool which respond to the butanol extract but not the phenol-extracted LPS. In contrast, none of the Lyb5- cells which are found in Xid CBA/n mice to phenol-extract LPS, indicating the that the Xid defect affects the differentiation of Lyb5- cells as well as the development of Lyb5+ B cells.